Glycosyl hydrolases represent a very diverse group of enzymes which hydrolyze glycosidic linkages between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate function.
Glycosyl hydrolases of particular industrial and technical interest are the α-amylases (E.C. 3. 2. 1. 1) which hydrolyze α-1,4-glycosidic linkages, which are located in the interior of polymers, of starch and starch-like polymers, such as, for example, amylose, amylopectin or glycogen, to form dextrins and β-1,6-branched oligosaccharides. They are among the most important of all the enzymes utilized in industry. There are two reasons for this: firstly they are usually, like many substrate-degrading enzymes, released by microorganisms into the surrounding medium so that they can be obtained on the industrial scale with comparatively little effort by fermentation and purification from the culture medium. Secondly, amylases are required for a wide range of applications.
The principal industrial use of α-amylase is in the production of glucose syrup. Other uses are, for example, that as active components in washing and cleaning compositions, for the treatment of raw materials in textile manufacture, for the production of adhesives or for the production of sugar-containing food products or food ingredients.
One example of an amylase which is employed particularly intensively in industry is the α-amylase from Bacillus licheniformis, which is supplied by Novozymes A/S, Bagsvaerd, Denmark, under the proprietary name Termamyl®. The amylase obtained from B. subtilis or B. amyloliquefaciens and disclosed in U.S. Pat. No. 1,227,374 is marketed by the same company under the name BAN®.
This amylase molecule, and its near relations, have been further developed in numerous inventions which were based on the object of optimizing, with the aid of various molecular biological modifications, their enzymatic properties for specific applications. Such optimizations may relate for example to the substrate specificities, the stability of the enzyme under various reaction conditions or the enzymatic activity itself. Examples of such optimizations which may be mentioned are the following property rights: EP 0410498 B1 for the sizing of textiles and WO 96/02633 A1 for the liquefaction of starch.
Since developments which consist merely of optimizations of only a few known starting enzymes are possibly restricted in the results which can be achieved, an intensive search is taking place in parallel thereto for comparable enzymes from other natural sources. Those which have been identified are starch-cleaving enzymes for example from Pimelobacter, Pseudomonas and Thermus for the manufacture of food products, cosmetics and drugs (EP 0 636 693 A2), ones of the same type from Rhizobium, Arthrobacter, Brevibacterium and Micrococcus (EP 0 628 630 A2), from Pyrococcus (WO 94/19454 A2) and Sulfolobus for the liquefaction of starch at high temperatures, or strongly acidic reaction conditions (EP 0 727 485 A1 and WO 96/02633 A1). Amylases for use at alkaline pH values have been found in Bacillus sp. (WO 95/26397 A1 and WO 97/00324 A1). Because of their low sensitivity to washing compositions, other amylases from various Bacilli (EP 0 670 367 A1) are suitable for use in washing or cleaning compositions.
Further optimizations of enzymes isolated from natural sources for the respective area of application can be carried out for example by molecular biological methods (for example as in U.S. Pat. No. 5,171,673 or WO 99/20768 A1) or by chemical modifications (DE 4013142 A1). The patent application WO 99/43793 A1, for example, describes a further development of the known Novamyl® α-amylase. Similarities in sequence between Novamyl® and known cyclodextrin glucanotransferases (CGTases) are utilized therein to construct, with the aid of molecular biology techniques, a multitude of related molecules. These comprise α-amylases with additional CGTase-specific consensus sequences (boxes) and functions or, conversely, CGTases with additional regions and functions typical of α-amylases, or chimeras of the two molecules. The point of this development is to optimize Novamyl® for these applications.
The new enzymes derived from the optimization of known glycosyl hydrolases and, in particular, amylases are, by their nature, limited in their properties because they represent local optima in the “fitness landscape” of glycosyl hydrolases. In order to find further, possibly even better, local optima it is necessary to start from other original enzymes which either themselves represent such a local optimum, or can be optimized with reasonable effort in the direction thereof. However, the finding of new glycosyltransferases which may be such original enzymes requires, by the route of classical microbiological screening, the isolation of defined strains. However, more than 95% of all occurring microorganisms cannot be cultivated, so that the new glycosyl hydrolases occurring in these microorganisms, including the amylases, have not to date been accessible to characterization.
α-Amylases are typically monomeric enzymes with a molecular weight of about 55 kDa. The central domain has an α/β barrel structure (TIM barrel) which, according to the current state of knowledge, represents the most widespread protein-folding motif. The linear protein strand consists of in each case 8 α-helices and 8 β-pleated sheet segments (β sheets) which occur in alternating sequence and fold three-dimensionally in such a way that the β-pleated sheets are disposed in the form of a circle in parallel orientation and are surrounded by the α-helices on the outside.
The enzymatic activity is probably derived from amino acid residues which are located at the C-terminal end of the individual helices and β-pleated sheets.
For the example of the amylase known as Termamyl from Bacillus licheniformis, the amino acid residues are the aspartate 231 (general base catalyst) and glutamate 261 (general acid catalyst). The residue Asp 328 which was originally regarded as general acid catalyst is, according to recent investigations, attributed with an essential influence on the electrostatic conditions in the active site. Further residues such as, for example, His 210 are thought to be involved in the binding of the substrate or further factors such as, for example, Ca2+ ions.
Besides the actual amylases, further enzymes are included in the amylase superfamily, for example α-1,6-glucosidases, cyclodextrin glucanotransferases (CGTases), isoamylases, neopullulanases, glycogen debranching enzyme, dextran glucosidases and glycosyltransferases.
The substrate binding specificity, which differs widely in some cases, is brought about by protein loops which are inserted between particular structural elements and form relatively small independent domains.
Thus, a B domain exists and is disposed between β-pleated sheets 3 and helix 3. It comprises in the case of α-amylases, and α-1,6-glucosidases and cyclodextrin glucanotransferases (CGTases) closely related thereto, approximately 40 amino acids, but may also, as, for example, in the case of glucan-debranching enzymes, comprise 270 amino acids (Jespersen, H. M.; E. A. MacGregor, B. Henrissat, M. Sierks and B. Svensson: “Starch- and Glycogen-Debranching and Branching Enzymes”, J. of Protein Chemistry, volume 12, pp. 791-805, 1993).
Further domains are disposed at the C terminus, such as the C domain in all enzymes and one or more further D or E domains (for example in CGTases). These are likewise attributed with involvement in substrate binding.
Despite this generally similar structure, the sequence homology of the linear protein strand varies widely, making it extremely difficult to define characteristic sequence segments as probes which could be used to search in an efficient manner for new amylases.
Structural elements responsible for the folding may also be formed without homology in the primary structure. Thus, the amylases from Bacillus licheniformis and Bacillus amyloliquefaciens are more than 80% identical. By contrast, the homology over all amylases of bacterial, animal and vegetable origin is less than 10% (Nakajima et al., Appl. Microbiol. Biotechnol., volume 23, pp. 355-360 (1986)).
Further TIM barrel proteins are, despite an identical protein-folding structure, no longer homologous in any way.
Thus, proteins having little similarity in terms of homology may nevertheless display an identical or closely related enzymic activity as long as there is a similar protein-folding pattern and the appropriate topological disposition, which is necessary for the catalytic reaction, of the amino acid residues involved.
It can be stated in summary that there are great problems in the finding of new glycosyl hydrolases both due to the impossibility of cultivating many microorganisms which produce glycosyl hydrolases of potential interest, and due to the often only small sequence homology within the family of glycosyl hydrolases.